The present invention relates to a process for making long chain wax esters biosynthetically. More particularly, the invention relates to the biosynthesis of wax esters by incubating a microorganism of the species Acinetobacter aerobically in the presence of ethanol as a primary food source.
Long chain wax esters are required in a variety of specialized industrial applications. One important application is in steel making, where a recently developed continuous casting process depends on continuous and reliable lubrication of the mold walls. U.S. Pat. No. 4,152,278 discloses lubricant compositions, comprising wax esters of fatty acids and alcohols, which are particularly advantageous in the continuous casting of steel. These wax esters contain either 0, 1, 2, 3 or 4 internally located carbon-carbon double bonds, with no more than 2 double bonds in either of the fatty acid or the fatty alcohol segments. Presently, these wax esters are derived from vegetable oil derivatives by a relatively complicated chemical synthesis involving reduction of long chain fatty acids to fatty alcohols, followed by esterification with fatty acids. Compositions prepared in this way are limited by the carbon chain length and by the degree of unsaturation in the original vegetable oil derivatives.
Also in the field of lubrication, there exists a need for synthetic, extreme pressure and antiwear lubricant additives. Such additives prevent destructive metal-to-metal contact in lubrication under high pressure and/or temperature conditions, such as occur in certain gear elements in automotive vehicles and in various industrial machines. General chemical properties which make wax esters useful as lubricant additives are carbon chain lengths of at least 14 carbons in both the fatty acid and fatty alcohol segments, and the presence of an internal carbon-carbon double bond in one or both of the segments.
In the past, the only known natural source of such lubricant-additive waxes has been the sperm whale, an endangered species. More recently, waxes derived from the jojoba plant have been used as lubricants and additives. The esters derived from jojoba oil are composed almost entirely of straight chain acids and alcohols, each segment being predominantly 20-22 carbon atoms in length and having one unsaturated bond. In laboratory testing, jojoba oil has been found comparable or superior to sperm oil as a lubricant additive. The disadvantage of this wax ester source is plant growth time--up to five years--between the time of planting and first harvesting of the waxes.
Microorganisms are a potential source of wax esters. Through metabolic action, wax esters can be produced from inexpensive, readily available hydrocarbons. For example, U.S. Pat. No. 3,409,506 describes the production of wax esters by Micrococcus cerificans (now referred to as Acinetobacter species HOl-N) from aliphatic hydrocarbon feed stocks. A characteristic of wax esters produced in this microbial system is that the principal wax esters will have a carbon chain length in the fatty acid and fatty alcohol segments equal to the chain length of the hydrocarbon used as a feed stock. This feature permits the fatty acid and fatty alcohol chain lengths in the wax ester to be controlled by the chain length of the feed stock used.
A major disadvantage of microbial production of wax esters from aliphatic hydrocarbon chain feed stocks, as reported in the prior art, is that saturated, rather than unsaturated, wax esters are formed. Above-cited U.S. Pat. No. 3,409,506 discloses that Micrococcus cerificans produces only cetyl palmitate saturated wax ester from hexadecane. Stewart et al., J. Bact. 78:726-730 (1959) state that Micrococcus cerificans produces only octadecyl stearate saturated wax ester from octadecane. Makula et al., J. Bact. 121:250-258 (1975) claim that only cetyl palmitate saturated wax ester is formed by Acinetobacter sp. HO1-N acting on hexadecane. Raymond et al., Adv. Appl. Microbiol. 14:93-121 (1971) and Krasilnikov et al., Mikrobiologiya 38:757-760 (1969) state the same finding (hexadecane going to cetyl palmitate wax ester only) for Nocardia species and Mycobacterium species, respectively.
Attempts have been made to produce wax esters containing carbon-carbon double bonds by feed stocks other than saturated hydrocarbons. The metabolic action of Acinetobacter sp. HO1-N (Makula et al., J. Bact. 121:250-258 (1975)) and 3 species of Acinetobacter (Gallagher, J. Gen. Microbiol. 68:245-247 (1971)) on amino acid feed stocks yielded unsaturated wax esters. Fixter et al., Biochem. Soc. Transl. 4:504-505 (1976), reported that the metabolic action of Acinetobacter species on acetate or succinate feed stocks yielded wax esters containing C.sub.14, C.sub.16 and C.sub.18 saturated and monounsaturated fatty acid and fatty alcohol segments. The metabolic action of Candida lipolytica on olefinic feed stocks yielded unsaturated wax esters (Stewart et al., Science 132:1254 (1960)). Both types of feed stocks yielding unsaturated wax esters are substantially more expensive than aliphatic hydrocarbons.
Guehler et al., Arch. Biochem. Biophys. 106:291-298 (1964) have reported the formation of wax esters by Euglena gracilis grown on a complex nutrient medium containing acetate and ethanol. The esters formed were saponified and the acids and alcohols and acids were converted into methyl esters and acetates, respectively. Analysis of these components by vapor phase chromotography showed a predominance of saturated C.sub.12, C.sub.13 and C.sub.14 linear chains in both the methyl ester and acetate fractions. Only small quantities of monounsaturated chains, including hexadecenoic acid (0.7%), heptadecenoic acid (0.1%) and octadecenoic acid (0.1%) were present.
One object of the present invention is to provide a method of producing, by microbial biosynthesis, wax esters having above-mentioned characteristics suitable for use as lubricants and lubricant additives.
Another object of the invention is to provide such a method which employs, as a microbial food source, inexpensive and readily available biomass-based feed stocks.
Yet another object of the invention is to provide such a method in which the degree of unsaturation of fatty acid and alcohol segments in the wax esters can be varied selectively by changing the temperature conditions employed in the method.
The invention includes a process for producing a mixture of wax esters having the general formula RCO.sub.2 R', where R and R' are each radicals selected from the group consisting of CH.sub.3 (CH.sub.2).sub.a CH.dbd.CH(CH.sub.2).sub.b --, where a+b equals 12-14 for R and 13-15 for R', and CH.sub.3 (CH.sub.2).sub.c --, where c equals 14-16 for R and 15-17 for R'. The process includes aerobically incubating a culture of microorganisms of the genus Acinetobacter in an aqueous mineral salts solution containing ethanol as a primary food source, and extracting the wax esters produced by the microorganisms.
In a preferred embodiment of the invention, the process includes selecting an incubation temperature known to produce, in such a wax esters mixture, a desired percentage of diene, monoene and saturated wax esters.
These and other objects and features of the invention will become more clearly understood by referring to the following description and claims.